Haploinsufficiency underlies the neurodevelopmental consequences of SLC6A1 variants.

Heterozygous variants in SLC6A1, encoding the GAT-1 GABA transporter, are associated with seizures, developmental delay, and autism. The majority of affected individuals carry missense variants, many of which are recurrent germline de novo mutations, raising the possibility of gain-of-function or dominant-negative effects. To understand the functional consequences, we performed an in vitro GABA uptake assay for 213 unique variants, including 24 control variants. De novo variants consistently resulted in a decrease in GABA uptake, in keeping with haploinsufficiency underlying all neurodevelopmental phenotypes. Where present, ClinVar pathogenicity reports correlated well with GABA uptake data; the functional data can inform future reports for the remaining 72% of unscored variants. Surface localization was assessed for 86 variants; two-thirds of loss-of-function missense variants prevented GAT-1 from being present on the membrane while GAT-1 was on the surface but with reduced activity for the remaining third. Surprisingly, recurrent de novo missense variants showed moderate loss-of-function effects that reduced GABA uptake with no evidence for dominant-negative or gain-of-function effects. Using linear regression across multiple missense severity scores to extrapolate the functional data to all potential SLC6A1 missense variants, we observe an abundance of GAT-1 residues that are sensitive to substitution. The extent of this missense vulnerability accounts for the clinically observed missense enrichment; overlap with hypermutable CpG sites accounts for the recurrent missense variants. Strategies to increase the expression of the wild-type SLC6A1 allele are likely to be beneficial across neurodevelopmental disorders, though the developmental stage and extent of required rescue remain unknown.

The full spectrum of SLC22 OCT1 mutations illuminates the bridge between drug transporter biophysics and pharmacogenomics.

Mutations in transporters can impact an individual's response to drugs and cause many diseases. Few variants in transporters have been evaluated for their functional impact. Here, we combine saturation mutagenesis and multi-phenotypic screening to dissect the impact of 11,213 missense single-amino-acid deletions, and synonymous variants across the 554 residues of OCT1, a key liver xenobiotic transporter. By quantifying in parallel expression and substrate uptake, we find that most variants exert their primary effect on protein abundance, a phenotype not commonly measured alongside function. Using our mutagenesis results combined with structure prediction and molecular dynamic simulations, we develop accurate structure-function models of the entire transport cycle, providing biophysical characterization of all known and possible human OCT1 polymorphisms. This work provides a complete functional map of OCT1 variants along with a framework for integrating functional genomics, biophysical modeling, and human genetics to predict variant effects on disease and drug efficacy.

Altered social cognition in early relapsing remitting multiple sclerosis.

INTRODUCTION: People with multiple sclerosis (pwMS) may suffer from some degree of impaired social cognition (SC), the process that integrates the mental operations underlying social interactions. SC is still not clearly characterized in the early stages of MS, and it is not defined whether SC is independent of cognitive impairment. METHODS: In this cross-sectional study, we aimed to compare SC measures in a population of early (≤5 years) relapsing-remitting MS (RRMS) with an age, sex, and education-matched control group. All participants performed a clinical and a comprehensive neuropsychological assessment. SC evaluation included assessment of facial emotion recognitionn by the Emotion Recognition Task, affective theory of mind (ToM) by the Reading the Mind in the eyes Test (RMET) and cognitive ToM by the Faux Pas test (FPT). Depression, anxiety, fatigue, and quality of life were also assessed. We included 38 pwMS (mean age 34.8 ± 8.7, 78.9% female sex, mean disease duration 1.9±1.3 years) and 38 healthy controls (mean age 34.9 ± 8.4, 81.6% female sex). RESULTS: Altered social cognition was present in 34.2% of pwMS. Participants with MS performed worse than controls on measures of cognitive ToM, and affective ToM. There were no differences regarding FER. Cognitive ToM and FER correlated with cognitive functions, but no correlation was found between affective ToM and cognitive tests. The only clinical factor associated with altered SC was poor quality of life. CONCLUSIONS: Social cognition impairment is already present in a significant percentage of early RRMS patients, namely ToM deficits. While cognitive ToM and FER appears to correlate with impaired cognitive results, affective ToM is likely independent of other cognitive functions.

The full spectrum of OCT1 (SLC22A1) mutations bridges transporter biophysics to drug pharmacogenomics.

Membrane transporters play a fundamental role in the tissue distribution of endogenous compounds and xenobiotics and are major determinants of efficacy and side effects profiles. Polymorphisms within these drug transporters result in inter-individual variation in drug response, with some patients not responding to the recommended dosage of drug whereas others experience catastrophic side effects. For example, variants within the major hepatic Human organic cation transporter OCT1 (SLC22A1) can change endogenous organic cations and many prescription drug levels. To understand how variants mechanistically impact drug uptake, we systematically study how all known and possible single missense and single amino acid deletion variants impact expression and substrate uptake of OCT1. We find that human variants primarily disrupt function via folding rather than substrate uptake. Our study revealed that the major determinants of folding reside in the first 300 amino acids, including the first 6 transmembrane domains and the extracellular domain (ECD) with a stabilizing and highly conserved stabilizing helical motif making key interactions between the ECD and transmembrane domains. Using the functional data combined with computational approaches, we determine and validate a structure-function model of OCT1s conformational ensemble without experimental structures. Using this model and molecular dynamic simulations of key mutants, we determine biophysical mechanisms for how specific human variants alter transport phenotypes. We identify differences in frequencies of reduced function alleles across populations with East Asians vs European populations having the lowest and highest frequency of reduced function variants, respectively. Mining human population databases reveals that reduced function alleles of OCT1 identified in this study associate significantly with high LDL cholesterol levels. Our general approach broadly applied could transform the landscape of precision medicine by producing a mechanistic basis for understanding the effects of human mutations on disease and drug response.

Characterization of P-glycoprotein orthologs from human, sheep, pig, dog, and cat.

The ATP-binding cassette transporter P-glycoprotein (P-gp) limits the oral bioavailability of many drugs. Although P-gp has been well studied in humans and mice, little is known about the substrate specificities of many of its species orthologs. To address this, we performed in vitro analysis of P-gp transporter function using HEK293 cells stably expressing human, ovine, porcine, canine, and feline P-gp. We also employed a human physiologically based pharmacokinetic (PBPK) model to assess variations in digoxin exposure resulting from altered P-gp function. Compared to human P-gp, sheep P-gp had significantly less digoxin efflux (2.3-fold ±0.04 vs. 1.8-fold ±0.03, p < .0001) and all species orthologs had significantly less quinidine efflux compared with human P-gp (p < .05). Human P-gp also had significantly greater efflux of talinolol compared to sheep and dog P-gp (1.9-fold ±0.04 vs. 1.6-fold ±0.06, p = .003 and 1.6-fold ±0.05, p = .0002, respectively). P-gp expression protected all lines against paclitaxel-induced toxicity, with sheep P-gp being significantly less protective. The inhibitor verapamil demonstrated dose-dependent inhibition of all P-gp orthologs. Finally, a PBPK model showed digoxin exposure was sensitive to altered P-gp activity. Overall, our study found that species differences in this major drug transporter exist and that the appropriate species ortholog of P-gp should be evaluated during veterinary drug development.

A Safe-by-Design Approach for the Synthesis of a Novel Cross-Linked Hyaluronic Acid with Improved Biological and Physical Properties.

Hyaluronic acid (HA) is a polymer with unique biological properties that has gained in interest over the years, with applications in pharmaceutical, cosmetic, and biomedical fields; however, its widespread use has been limited by its short half-life. Therefore, a new cross-linked hyaluronic acid was designed and characterized using a natural and safe cross-linking agent, such as arginine methyl ester, which provided improved resistance to enzymatic action, as compared to the corresponding linear polymer. The antibacterial profile of the new derivative was shown to be effective against S. aureus and P. acnes, making it a promising candidate for use in cosmetic formulations and skin applications. Its effect on S. pneumoniae, combined with its excellent tolerability profile on lung cells, also makes this new product suitable for applications involving the respiratory tract.

The development of a 3D-printed in vitro integrated oro-pharyngeal air-liquid interface cellular throat model for drug transport.

To simulate the deposition of drugs in the oro-pharynx region, several in vitro models are available such as the United States Pharmacopeia-Induction Port (USP-IP) throat and the Virginia Commonwealth University (VCU) models. However, currently, there is no such in vitro model that incorporates a biological barrier to elucidate drug transport across the pharyngeal cells. Cellular models such as in vitro air-liquid interface (ALI) models of human respiratory epithelial cell lines are extensively used to study drug transport. To date, no studies have yet been performed to optimise the ALI culture conditions of the human pharyngeal cell line Detroit 562 and determine whether it could be used for drug transport. Therefore, this study aimed to develop a novel 3D-printed throat model integrated with an ALI cellular model of Detroit 562 cells and optimise the culture conditions to investigate whether the combined model could be used to study drug transport, using Lidocaine as a model drug. Differentiating characteristics specific to airway epithelia were assessed using 3 seeding densities (30,000, 60,000, and 80,000 cells/well (c/w), respectively) over 21 days. The results showed that Detroit 562 cells completely differentiates on day 18 of ALI for both 60,000 and 80,000 c/w with significant mucus production, showing response to bacterial and viral stimuli and development of functional tight junctions and Lidocaine transport with no significant differences observed between the ALI models with the 2 cell seeding densities. Results showed the suitability of the Low density (60,000 c/w or 1.8 × 105 cells/cm2) ALI model to study drug transport. Importantly, the developed novel 3D-printed throat model integrated with our optimised in vitro Detroit 562 ALI model showed transport of Lidocaine throat spray. Overall, the study highlights the potential of the novel 3D-printed bio-throat integrated model as a promising in vitro system to investigate the transport of inhalable drug therapies targeted at the oro-pharyngeal region.

In vitro and in vivo applications of a universal and synthetic thermo-responsive drug delivery hydrogel platform.

A synthetic and thermo-responsive polymer, poly(N-isopropylacrylamide)-co-(polylactide/2-hydroxy methacrylate)-co-(oligo (ethylene glycol)), is used to formulate a universal carrier platform for sustained drug release. The enabling carrier, denoted as TP, is prepared by dissolving the polymer in an aqueous solution at a relatively neutral pH. A wide range of therapeutic moieties can be incorporated without the need for the addition of surfactants, organic solvents, and other reagents to the carrier system. The resulting solution is flowable through fine gauge needle, allowing accurate administration of TP to the target site. After injection, TP carrier undergoes a coil to globe phase transition to form a hydrogel matrix at the site. The benign nature of the polymer carrier and its physical gelation process are essential to preserve the biological activity of the encapsulated compounds while the adhesive hydrogel nature of the matrix allows sustained elusion and controlled delivery of the incorporated therapeutics. The TP carrier system has been shown to be non-toxic and elicits a minimal inflammatory response in multiple in vitro studies. These findings suggest the suitability of TP as an enabling carrier of therapeutics for localized and sustained drug delivery. To confirm this hypothesis, the capabilities of TP to encapsulate and effectively deliver multiple therapeutics of different physicochemical characteristics was evaluated. Specifically, a broad range of compounds were tested, including ciprofloxacin HCl, tumor necrosis factor-alpha (TNF-α), transforming growth factor beta 1 (TGF-ß1), and recombinant human bone morphogenetic protein 2 (BMP2). In vitro studies confirmed that TP carrier is able to control the release of the encapsulated drugs over an extended period of time and mitigate their burst release regardless of the compounds' physiochemical properties for the majority of the loaded therapeutics. Importantly, in vitro and in vivo animal studies showed that the released drugs from the TP hydrogel matrix remained potent and bioactive, confirming the high potential of the TP polymer system as an enabling carrier.

Describing inhibitor specificity for the amino acid transporter LAT1 from metainference simulations.

The human L-type amino acid transporter 1 (LAT1; SLC7A5) is a membrane transporter of amino acids, thyroid hormones, and drugs such as the Parkinson's disease drug levodopa (L-Dopa). LAT1 is found in the blood-brain barrier, testis, bone marrow, and placenta, and its dysregulation has been associated with various neurological diseases, such as autism and epilepsy, as well as cancer. In this study, we combine metainference molecular dynamics simulations, molecular docking, and experimental testing, to characterize LAT1-inhibitor interactions. We first conducted a series of molecular docking experiments to identify the most relevant interactions between LAT1's substrate-binding site and ligands, including both inhibitors and substrates. We then performed metainference molecular dynamics simulations using cryoelectron microscopy structures in different conformations of LAT1 with the electron density map as a spatial restraint, to explore the inherent heterogeneity in the structures. We analyzed the LAT1 substrate-binding site to map important LAT1-ligand interactions as well as newly described druggable pockets. Finally, this analysis guided the discovery of previously unknown LAT1 ligands using virtual screening and cellular uptake experiments. Our results improve our understanding of LAT1-inhibitor recognition, providing a framework for rational design of future lead compounds targeting this key drug target.

Thermoresponsive and Injectable Hydrogel for Tissue Agnostic Regeneration.

Injectable hydrogels can support the body's innate healing capability by providing a temporary matrix for host cell ingrowth and neovascularization. The clinical adoption of current injectable systems remains low due to their cumbersome preparation requirements, device malfunction, product dislodgment during administration, and uncontrolled biological responses at the treatment site. To address these challenges, a fully synthetic and ready-to-use injectable biomaterial is engineered that forms an adhesive hydrogel that remains at the administration site regardless of defect anatomy. The product elicits a negligible local inflammatory response and fully resorbs into nontoxic components with minimal impact on internal organs. Preclinical animal studies confirm that the engineered hydrogel upregulates the regeneration of both soft and hard tissues by providing a temporary matrix to support host cell ingrowth and neovascularization. In a pilot clinical trial, the engineered hydrogel is successfully administered to a socket site post tooth extraction and forms adhesive hydrogel that stabilizes blood clot and supports soft and hard tissue regeneration. Accordingly, this injectable hydrogel exhibits high therapeutic potential and can be adopted to address multiple unmet needs in different clinical settings.

Protective Abilities of an Inhaled DPI Formulation Based on Sodium Hyaluronate against Environmental Hazards Targeting the Upper Respiratory Tract.

The exposure of lung epithelium to environmental hazards is linked to several chronic respiratory diseases. We assessed the ability of an inhaled dry powder (DPI) medical device product (PolmonYDEFENCE/DYFESATM, SOFAR SpA, Trezzano Rosa, Italy), using a formulation of sodium hyaluronate (Na-Hya) as the key ingredient as a defensive barrier to protect the upper respiratory tract. Specifically, it was evaluated if the presence of the barrier formed by sodium hyaluronate present on the cells, reducing direct contact of the urban dust (UD) with the surface of cells can protect them in an indirect manner by the inflammatory and oxidative process started in the presence of the UD. Cytotoxicity and the protection capability against the oxidative stress of the product were tested in vitro using Calu-3 cells exposure to UD as a trigger for oxidative stress. Inflammation and wound healing were assessed using an air-liquid interface (ALI) culture model of the Calu-3 cells. Deposition studies of the formulation were conducted using a modified Anderson cascade impactor (ACI) and the monodose PillHaler® dry powder inhaler (DPI) device, Na-Hya was detected and quantified using high-performance-liquid-chromatography (HPLC). Solubilised PolmonYDEFENCE/DYFESATM gives protection against oxidative stress in Calu-3 cells in the short term (2 h) without any cytotoxic effects. ALI culture experiments, testing the barrier-forming (non-solubilised) capabilities of PolmonYDEFENCE/DYFESATM, showed that the barrier layer reduced inflammation triggered by UD and the time for wound closure compared to Na-Hya alone. Deposition experiments using the ACI and the PillHaler® DPI device showed that the majority of the product was deposited in the upper part of the respiratory tract. Finally, the protective effect of the product was efficacious for up to 24 h without affecting mucus production. We demonstrated the potential of PolmonYDEFENCE/DYFESATM as a preventative barrier against UD, which may aid in protecting the upper respiratory tract against environmental hazards and help with chronic respiratory diseases.

Understanding the effects of aerodynamic and hydrodynamic shear forces on Pseudomonas aeruginosa biofilm growth.

Biofilms are communities of bacterial cells encased in a self-produced polymeric matrix and exhibit high tolerance towards environmental stress. Despite the plethora of research on biofilms, most biofilm models are produced using mono-interface culture in static flow conditions, and knowledge of the effects of interfaces and mechanical forces on biofilm development remains fragmentary. This study elucidated the effects of air-liquid (ALI) or liquid-liquid (LLI) interfaces and mechanical shear forces induced by airflow and hydrodynamic flow on biofilm growing using a custom-designed dual-channel microfluidic platform. Results from this study showed that comparing biofilms developed under continuous nutrient supply and shear stresses free condition to those developed with limited nutrient supply, ALI biofilms were four times thicker, 60% less permeable, and 100 times more resistant to antibiotics, while LLI biofilms were two times thicker, 20% less permeable, and 100 times more resistant to antibiotics. Subjecting the biofilms to mechanical shear stresses affected the biofilm structure across the biofilm thickness significantly, resulting in generally thinner and denser biofilm compared to their controlled biofilm cultured in the absence of shear stresses, and the ALI and LLI biofilm's morphology was vastly different. Biofilms developed under hydrodynamic shear stress also showed increased antibiotic resistance. These findings highlight the importance of investigating biofilm growth and its mechanisms in realistic environmental conditions and demonstrate a feasible approach to undertake this study using a novel platform.

The Outcome of Patients with Amyloid-Negative Amnestic Mild Cognitive Impairment.

BACKGROUND: Patients with amnestic mild cognitive impairment (aMCI) are usually at an initial stage of Alzheimer's disease (AD). However, some patients with aMCI do not present biomarkers of amyloid pathology characteristic of AD. The significance of amyloid-negative aMCI is not presently clear. OBJECTIVE: To know the etiology and prognosis of amyloid-negative aMCI. METHODS: Patients who fulfilled criteria for aMCI and were amyloid negative were selected from a large cohort of non-demented patients with cognitive complaints and were followed with clinical and neuropsychological assessments. RESULTS: Few amyloid-negative aMCI had evidence of neurodegeneration at the baseline, as reflected in cerebrospinal fluid elevated tau protein levels. About half of the patients remained essentially stable for long periods of time. Others manifested a psychiatric disorder that was not apparent at baseline, namely major depression or bipolar disorder. Remarkably, about a quarter of patients developed neurodegenerative disorders other than AD, mostly frontotemporal dementia or Lewy body disease. CONCLUSION: Amyloid-negative aMCI is a heterogeneous condition. Many patients remain clinically stable, but others may later manifest psychiatric conditions or evolve to neurodegenerative disorders. Prudence is needed when communicating to the patient and family the results of biomarkers, and clinical follow-up should be advised.

An adaptable microreactor to investigate the influence of interfaces on Pseudomonas aeruginosa biofilm growth.

Biofilms are ubiquitous and notoriously difficult to eradicate and control, complicating human infections and industrial and agricultural biofouling. However, most of the study had used the biofilm model that attached to solid surface and developed in liquid submerged environments which generally have neglected the impact of interfaces. In our study, a reusable dual-chamber microreactor with interchangeable porous membranes was developed to establish multiple growth interfaces for biofilm culture and test. Protocol for culturing Pseudomonas aeruginosa (PAO1) on the air-liquid interface (ALI) and liquid-liquid interface (LLI) under static environmental conditions for 48 h was optimized using this novel device. This study shows that LLI model biofilms are more susceptible to physical disruption compared to ALI model biofilm. SEM images revealed a unique "dome-shaped" microcolonies morphological feature, which is more distinct on ALI biofilms than LLI. Furthermore, the study showed that ALI and LLI biofilms produced a similar amount of extracellular polymeric substances (EPS). As differences in biofilm structure and properties may lead to different outcomes when using the same eradication approaches, the antimicrobial effect of an antibiotic, ciprofloxacin (CIP), was chosen to test the susceptibility of a 48-h-old P. aeruginosa biofilms grown on ALI and LLI. Our results show that the minimum biofilm eradication concentration (MBEC) of 6-h CIP exposure for ALI and LLI biofilms is significantly different, which are 400 µg/mL and 200 µg/mL, respectively. These results highlight the importance of growth interface when developing more targeted biofilm management strategies, and our novel device provides a promising tool that enables manipulation of realistic biofilm growth. KEY POINTS: • A novel dual-chamber microreactor device that enables the establishment of different interfaces for biofilm culture has been developed. • ALI model biofilms and LLI model biofilms show differences in resistance to physical disruption and antibiotic susceptibility.

Design, Synthesis, Characterization, and In Vitro Evaluation of a New Cross-Linked Hyaluronic Acid for Pharmaceutical and Cosmetic Applications.

Hyaluronic acid (HA), an excellent biomaterial with unique bio properties, is currently one of the most interesting polymers for many biomedical and cosmetic applications. However, several of its potential benefits are limited as it is rapidly degraded by hyaluronidase enzymes. To improve the half-life and consequently increase performance, native HA has been modified through cross-linking reactions with a natural and biocompatible amino acid, Ornithine, to overcome the potential toxicity commonly associated with traditional linkers. 2-chloro-dimethoxy-1,3,5-triazine/4-methylmorpholine (CDMT/NMM) was used as an activating agent. The new product (HA-Orn) was extensively characterized to confirm the chemical modification, and rheological analysis showed a gel-like profile. In vitro degradation experiments showed an improved resistance profile against enzymatic digestions. Furthermore, in vitro cytotoxicity studies were performed on lung cell lines (Calu-3 and H441), which showed no cytotoxicity.

Regeneration of critical-sized defects, in a goat model, using a dextrin-based hydrogel associated with granular synthetic bone substitute.

The development of injectable bone substitutes (IBS) have obtained great importance in the bone regeneration field, as a strategy to reach hardly accessible defects using minimally invasive techniques and able to fit to irregular topographies. In this scenario, the association of injectable hydrogels and bone graft granules is emerging as a well-established trend. Particularly, in situ forming hydrogels have arisen as a new IBS generation. An in situ forming and injectable dextrin-based hydrogel (HG) was developed, aiming to act as a carrier of granular bone substitutes and bioactive agents. In this work, the HG was associated to a granular bone substitute (Bonelike®) and implanted in goat critical-sized calvarial defects (14 mm) for 3, 6 and 12 weeks. The results showed that HG improved the handling properties of the Bonelike® granules and did not affect its osteoconductive features, neither impairing the bone regeneration process. Human multipotent mesenchymal stromal cells from the umbilical cord, extracellular matrix hydrolysates and the pro-angiogenic peptide LLKKK18 were also combined with the IBS. These bioactive agents did not enhance the new bone formation significantly under the conditions tested, according to micro-computed tomography and histological analysis.

Memory awareness in patients with Major Depressive Disorder.

BACKGROUND: Subjective Memory Complaints (SMC) along with cognitive deficits are frequently observed in patients with Major Depressive Disorder (MDD). The relationship between SMC and objective memory performance in patients with MDD was evaluated, in comparison with patients with Mild Cognitive Impairment due to Alzheimer's Disease (MCI-AD) and healthy controls (HC). METHODS: Patients with MDD (n = 47), MCI-AD (n = 43) and HC (n = 45) were assessed with a self-report memory complaints scale (SMCS) and underwent a comprehensive clinical and neuropsychological assessment. A discrepancy score between the Logical Memory delayed recall and the SMCS total score was calculated as a measure of memory awareness. RESULTS: Patients with MDD (12.5 ± 4.4) and patients with MCI-AD (10.9 ± 4.1) had not significantly different SMCS total scores, whereas HC showed significantly lower scores (4.0 ± 3.0). As much as 74.5% of patients with MDD patients and 65.1% of patients with MCI-AD reported prominent memory complaints, whereas only 4.4% of HC did. Patients with MDD had relatively preserved memory tests, resulting in a higher discrepancy score than both patients with MCI-AD and HC. The SMCS total score correlated positively with depressive symptoms in the 3 groups of participants. CONCLUSIONS: Patients with MDD showed inaccurate memory self-awareness as they under-estimated their memory functioning, a pattern distinct from both patients with MCI-AD and HC.

Co-delivery of inhalable therapies: Controlling active ingredients spatial distribution and temporal release.

The management of respiratory diseases relies on the daily administration of multiple active pharmaceutical ingredients (APIs), leading to a lack of patient compliance and impaired quality of life. The frequency and dosage of the APIs result in increased side effects that further worsens the overall patient condition. Here, the manufacture of polymer-polymer core-shell microparticles for the sequential delivery of multiple APIs by inhalation delivery is reported. The microparticles, composed of biodegradable polymers silk fibroin (shell) and poly(L-lactic acid) (core), incorporating ciprofloxacin in the silk layer and ibuprofen (PLLA core) as the antibiotic and anti-inflammatory model APIs, respectively. The polymer-polymer core-shell structure and the spatial distribution of the APIs have been characterized using cutting-edge synchrotron macro ATR-FTIR technique, which was correlated with the respective API sequential release profiles. The APIs microparticles had a suitable size and aerosol properties for inhalation therapies (≤4.94 ± 0.21µm), with low cytotoxicity and immunogenicity in healthy lung epithelial cells. The APIs compartmentalization obtained by the microparticles not only could inhibit potential actives interactions but can provide modulation of the APIs release profiles via an inhalable single administration.